Toxic cocktails in soils - Evidence for synergistic effects of the imidacloprid-epoxiconazole mixture on earthworm life-history traits

Abstract Soils are vital reservoirs of biodiversity and providers of ecosystem services, yet they are increasingly threatened by agricultural intensification and pesticide use. Residues often persist as complex mixtures, while environmental risk assessment still largely focuses on single substances, potentially underestimating mixture effects. Earthworms play a key role in soil functioning and are particularly vulnerable to pesticide contamination. We investigated the effects of a binary mixture of epoxiconazole and imidacloprid, two persistent and frequently detected pesticides, on life-history traits of Aporrectodea caliginosa . We estimated each compound relative potency using dose–response experiments on juvenile growth and cocoon production. Next, we assessed the potential for synergy or antagonism in a fixed-ratio ray design including five concentration ratios and seven additive isoboles (36 conditions). Both compounds showed significant toxicity. Imidacloprid showed high potency (juvenile growth NOEC = 0.28 mg/kg; reproduction EC 50 = 0.55 mg/kg), whereas epoxiconazole had moderate effects (juvenile growth NOEC = 9.3 mg/kg; reproduction EC 50 = 126.8 mg/kg). Reproductive endpoints were more sensitive than adult growth, with juvenile growth being the most sensitive overall. Mixture analysis using Jonker’s models revealed significant deviation from Independent Action only under the simple interaction model, indicating synergism, consistent with cytochrome P450 interference reported in other taxa. Field-reported imidacloprid concentrations often approach effect thresholds, suggesting potential risks for earthworm populations. Overall, the combined effects of epoxiconazole and imidacloprid may exceed predictions not taking interactions into account. These results highlight the need to incorporate pesticide mixture effects into environmental risk assessment. Environmental Implications Pesticide residues persist in agricultural soils as complex mixtures, yet risk assessment still focuses mainly on single substances. This study shows that the combined effects of imidacloprid and epoxiconazole on earthworm reproduction can exceed predictions based on Independent Action, with evidence of synergistic interactions. Effect thresholds for imidacloprid approach reported maximum environmental concentrations, indicating limited safety margins for soil organisms. These findings suggest that mixture exposures may pose greater ecological risks than currently anticipated and highlight the need to integrate pesticide mixture toxicity and potential synergism into environmental risk assessment frameworks. Graphical abstract